Bottom Line:
Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex.We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling.Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. rsiegel@nih.gov

ABSTRACTFas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

fig3: Differential coclustering of SPOTS with lipid rafts in type I and type II cells. Two-color fluorescence microscopy of cells treated with anti-Fas in the presence of Alexa-594–labeled cholera toxin B (red) for 30 min and then stained for Fas with anti-IgG3 Alexa-488 (green). Arrowheads mark examples of membrane areas with bright patches of Fas and the CTB staining at the same location.

Mentions:
Lipid raft microdomains can facilitate oligomerization of receptors into larger signaling complexes. Therefore, we investigated the relationship between SPOTS and lipid rafts in different cell types. When lipid rafts were imaged with cholera-toxin B after Fas receptor cross-linking, we observed that lipid raft microdomains coclustered with SPOTS in type I cells (Fig. 3). In type II cells, SPOTS formed without coclustering of the cholera-toxin label, suggesting that lipid rafts remained diffusely distributed on the plasma membrane. This data agrees with previous findings that Fas signaling in type-II cells is independent of lipid rafts (Muppidi and Siegel, 2004).

fig3: Differential coclustering of SPOTS with lipid rafts in type I and type II cells. Two-color fluorescence microscopy of cells treated with anti-Fas in the presence of Alexa-594–labeled cholera toxin B (red) for 30 min and then stained for Fas with anti-IgG3 Alexa-488 (green). Arrowheads mark examples of membrane areas with bright patches of Fas and the CTB staining at the same location.

Mentions:
Lipid raft microdomains can facilitate oligomerization of receptors into larger signaling complexes. Therefore, we investigated the relationship between SPOTS and lipid rafts in different cell types. When lipid rafts were imaged with cholera-toxin B after Fas receptor cross-linking, we observed that lipid raft microdomains coclustered with SPOTS in type I cells (Fig. 3). In type II cells, SPOTS formed without coclustering of the cholera-toxin label, suggesting that lipid rafts remained diffusely distributed on the plasma membrane. This data agrees with previous findings that Fas signaling in type-II cells is independent of lipid rafts (Muppidi and Siegel, 2004).

Bottom Line:
Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex.We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling.Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

Affiliation:
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. rsiegel@nih.gov

ABSTRACTFas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.